Tag Archives: bacterial challenge

All dilutions were performed with 1x LB+ 1%NaCl. 100uL were plated of all dilutions (see below) on 1xLB+1%NaCL plates. Plates were incubated O/N @ 37C. Colonies will be counted tomorrow to determine CFU for each sample.

Plated 100uL of:

V.vulnificus, t=0, 1:1,000,000 and 1:10,000,000

V.vulnificus H2O sample, t=1 & 3, 1:10,000 and 1:1,000,000

V.tubiashii, t=0, 1:1,000,000

Control H2O sample, t=1 & 3, Undiluted

Samples tubes containing bacteria and dilutions were stored @ 4C.

UPDATE 20110112:

Colony counts and calculations

V.vulnificus 0hr = Both dilutions produced a total lawn of bacteria. Uncountable. Will plate higher dilutions, but this will now only be a rough estimate due to the time that has passed.

—UPDATE—

New serial dilutions (90uL plated) of V.vulnificus 0hr were performed, down to 10^12. The only countable plate was the 10^12 dilution.

400mL O/N culture (1x LB+1% NaCl, 37C, 150RPM, 1L flask) of V.vulnificus (STRAIN??) and V.tubiashii (Strain: RE22) were pelleted (4300RPM, 25C, Sorvall ST-H750 rotor). Supe was removed and pellets were each resuspended in 50mL sea water. 1mL was taken from each to use for dilutions to determine colony forming units (CFU).

Two containers were set up with each containing 16 C.gigas, and air stone and 8L of sea water. The entire 50mL of V.vulnificus was added to one of the containers. 8 oysters were sampled (gill and mantle tissue) from each container at 1hr and 3hrs after the addition of V.vulnificus culture and immediately frozen on dry ice. Samples were stored @ -80C in the “Gigas Vibrio Exposure 1,3hrs 1/11/11″ box. Additionally, 1mL samples of the water were taken at each time to determine CFU in the water.

In addition to the samples taken above, the following tissues were taken from 5 control oysters at the 3hr time point and treated/stored in the same fashion as the others, specifically for assessment of cyclooxygenase tissue distribution analysis: muscle, digestive gland/gonad (difficult to differentiate)

100uL of a 1:1,000,000 dilution of the control bacteria were plated on 1x LB + 1% NaCl plates and incubated O/N @ RT. 100uL of a 1:10,000,000 dilution of the exposed bacteria were plated on 1x LB + 1% NaCl plates and incubated O/N @ RT. Control colony count the next day = 563 colony forming units (CFU). Exposed colony count the next day = 367 CFU.